A primary feature of such pathologies as inflammation and autoimmune diseases is the accumulation of activated leukocytes in affected tissues. The process by which leukocytes transmigrate from the circulation at a site of inflammation involves a cascade of interactions that can be divided into four major steps: tethering and rolling, activation, firm adhesion, and transmigration (Springer, T., Ann. Rev. Physiol., 57:827 (1995)). Initially, leukocytes are lightly tethered to the endothelium and roll along its surface. This is followed by cell activation, mediated by soluble chemotactic stimuli, which initiates the development of a firmer bond between individual leukocytes and endothelial cells. The firm bond then results in the successful adhesion and transmigration of the leukocytes through endothelial cell junctions. The steps occur in series and each is essential for transmigration to occur. This also means that transmigration can be modulated at each step, thus providing a number of potential targets for pharmacological inhibition.
The receptors involved in leukocyte migration have, to a large extent, been characterized as belonging to particular cell adhesion molecule families (Carlos and Harlan, Blood 84:2068 (1994)). The initial attachment and rolling step is mediated by a family of adhesion receptors referred to as selecting. Firm adhesion is mediated by interaction of leukocyte surface integrins with molecules of the immunoglobulin superfamily expressed on the surface of the endothelium. Both integrins and the immunoglobulin-type adhesion molecules are also primarily involved in leukocyte transmigration. After transmigration, the leukocytes rely on integrins to traverse through the extracellular matrix and remain at the site of inflammation.
Integrins are a large family of heterodimeric glycoproteins composed of two noncovalently associated subunits, α and β (Hynes, R, Cell, 69:11 (1992)). There are at least 16 different α subunits (α1–α9αL, αM, αD, αX, αE, αIIb, αv) and at least 9 different β (β1–β9) subunits. Integrins are divided into sub-families, based upon the β subunit. Leukocytes express a number of different integrins, including α4β1, α5β1, α6β1, α4β7, αLβ2, αXβ2, and αVβ3.
α4β1 integrin, also known as very late antigen-4 (VLA-4) or CD49d/CD29, is expressed on monocytes, lymphocytes, eosinophils, and basophils, all of which are key effector cells in various inflammatory disorders (Helmer, M., Ann. Rev. Immunol., 8:365 (1990)). α4β1 integrin serves as a receptor for vascular cell adhesion molecule-1 (VCAM-1), as well as to the extracellular protein fibronectin (FN) (Elices et al., Cell, 60:577 (1990)). Anti-inflammatory effects and delayed disease progression have been demonstrated after in vivo monoclonal antibody blockade of the α4β1/VCAM-1 pathway (Lobb et al., J. Clin. Invest., 94:1722–28 (1994)). In a guinea pig model of pulmonary inflammation, anti-α4 inhibited both antigen-induced bronchial hyperreactivity and leukocyte recruitment in bronchoalveolar lavage fluid (Pretolani et al., J. Exp. Med. 180:795 (1994)). Antibodies to α4 or VCAM-1, prevented antigen-induced eosinophil infiltration of the mouse trachea (Nakajima et al., J. Exp. Med. 179:1145 (1994)). α4 or VCAM-1 monoclonal antibody treatment also delayed or prevented cutaneous delayed hypersensitivity response in mice and monkeys (Chisholm et al., Eur. J. Immunol. 23:682 (1993); Silber et al., J. Clin. Invest., 93:1554 (1993); cardiac allograft rejection in mice, accompanied by specific immunosuppression (Isobe et al., J. Immunol., 153:5810 (1994); graft-versus-host disease in mice after bone marrow transfer (Yang et al., Proc. Natl. Acad. Sci. USA, 90:10494, (1993); and experimental autoimmune encephalomyelitis in rats and mice (Yednock et al., Nature, 356:63 (1992);. Baron et al., J. Exp. Med. 177:57 (1993)).
Rational drug design studies have produced soluble VCAM-Ig fusion protein containing the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region. In vivo administration of the fusion protein significantly delays the onset of adoptively transferred autoimmune diabetes in nonobese diabetic mice (Jakubowski et al., J. Immunol., 155:938 (1995)). Another approach has used three-dimensional crystallographic structures of VCAM-1 fragments to synthesize cyclic peptide antagonists that closely mimicked the α4 integrin binding loop in domain 1 of VCAM-1. Synthetic VCAM-1 peptide CQIDSPC, was able to inhibit the adhesion of VLA-4-expressing cells to purified VCAM-1 (Wang et al., Proc. Natl. Acad. Sci. USA. 92:5714 (1995)).
An additional strategy is to block the binding of α4β1 to its other counter receptor, that is, an alternatively spliced region of fibronectin containing the connecting segment-1 (CS-1) motif (E. A. Wayner, J. Cell. Biol., 116:489 (1992)). A synthetic CS-1 tetrapeptide (phenylacetic acid-Leu-Asp-Phe-d-Pro-amide) inhibited VLA-4-mediated lymphocyte adherence in vitro and reduced accelerated coronary arteriopathy in rabbit cardiac allografts (Molossi et al., J. Clin. Invest., 95:2601 (1995)). Each of these studies provide evidence that selective inhibition of α4β1/VCAM-1 mediated adhesion is a proven strategy in the treatment of autoimmune and allergic inflammatory diseases.
Moreover, while U.S. Pat. No. 5,821,231 and PCT Applications WO 96/22966, WO 97/03094, WO 98/04247 and WO 98/04913 describe compounds exhibiting VLA-4 inhibitory activity in in vitro binding assays, none of the described compounds have exhibited efficacy in oral administration.
Accordingly, despite these advances, there remains a need for small, non-peptidic, specific inhibitors of VLA-4 dependent cell adhesion that are orally bioavailable and that are suitable for the long-term treatment of chronic inflammatory diseases and other pathologies associated with leukocyte migration and adhesion.